Adenovirus vectors (Advectors) designed to express an antigenic gene have shown great promise as vaccines. Although GenVec and other groups have constructed numerous Advectors expressing a variety of antigens a substantial subset of antigen genes have been found to be inhibitory to Advector construction and production. This limitation was resolved in our phase I research by blocking antigen expression with a repressor system that is over 50-fold stronger than the commonly used TetR system. The technology was demonstrated to be applicable to multiple serotype groups of Advectors. In this phase 2 SBIR grant application we propose to use our optimized repressor to make a production cell line that is broadly applicable to vaccine vectors based on different human serotype groups and encoding inhibitory antigens. At the end of this phase 2 research a cell line certified ready for use in GMP banking will be available. No such cell line presently exists. The strategy to make the preferred repressor cell line is described in the three aims of this proposal. In Aim 1 the preferred repressor cell line with an optimized expression cassette will be constructed. Development of an optimized expression cassette is required to circumvent the epigenetic silencing found with the expression cassette used in our phase 1 research. In Aim 2 the preferred repressor cell line will be identified and certified for use in GMP banking. Since it is anticipated that Advector vaccines will be produced in suspension cells for commercial purposes, in Aim 3 we will demonstrate that the preferred repressor cell line can be adapted to grow in suspension and support production of Advectors from multiple serotype groups with inhibitory antigens. The final GMP manufacturing cell line will speed and facilitate the testing of Advector vaccine concepts for many antigens, hasten their progress to clinical testing and reduce the cost of goods for future approved vaccine products. PUBLIC HEALTH RELEVANCE: Adenovirus vector-based vaccines show great promise for protecting against many human diseases. However, some promising vaccines can not be made. In this proposal we overcome this limitation by developing a cell line that ensures any Adenovirus vector vaccine can be produced.